Common methods of analyzing gene function involve either knocking out gene expression and corresponding gene function, or over-expressing a gene and looking for an associated phenotype.
Conventional mutagenesis techniques frequently result in the identification of loss-of-function mutants and associated gene mutations that interfere with the native gene. However, eukaryotic genomes contain a significant number of functional genes that have redundant coding sequences and regulatory regions within the genome. In addition, such methods do not often result in the identification of genes where loss-of-function results in early lethality. Both of these categories may potentially be identified through a method that results in gain-of-function.
Gain-of-function mutants may result from various mutations in a coding sequence that effect constitutive activation of the resulting protein, or by mutations that alter the level or pattern of gene expression. The latter type of mutations may be the result of altered promoter function in terms of the level of expression, for example, a constitutive versus inducible promoter, tissue or developmental stage specificity of a promoter or other regulatory element or enhanced native promoter activity.
Activation tagging is a method by which genes are randomly and strongly upregulated on a genome-wide scale, after which specific phenotypes can be screened for and selected. Activation tagging is the insertion of transcriptional enhancers randomly throughout a genome in order to increase the transcriptional activity of genes linked to the site of enhancer insertion and/or to down-regulate or inhibit production of functional transcripts from transcription units (coding sequence and regulatory sequences) in which the enhancer has inserted. The transcriptional enhancers may insert near genes, up-regulate their transcription and thereby create altered phenotypes. Lines are considered to be “tagged” because in any individual line the site where the enhancer integrates can be determined and the presence of the enhancer can be associated with a mutant phenotype by genetic analysis.
Activation tagging has been used to activate genes in a variety of plants. An activation T-DNA tagging construct was used to activate genes in tobacco cell culture allowing the cells to grow in the absence of plant growth hormones (Walden et al., Plant Mol. Biol. 26: 1521-1528, 1994). A series of publications followed, including reports of genes isolated from plant genomic sequences flanking the T-DNA tag and putatively involved in plant growth hormone responses. (See, e.g., Miklashevichs et al., Plant J. 12: 489-498, 1997; Harling et al., EMBO J. 16: 5855-5866, 1997; Walden et. al., EMBO J. 13: 4729-4736, 1994 and Schell et al., Trends Plant Sci. 3: 130, 1998 which discusses investigation of a group of related studies.) In a similar study in Arabidopsis, a single gene was isolated from plant genomic DNA by plasmid rescue, identified and found to contain a gene, CKI1, which has been implicated in cytokinin responses in plants, the phenotype of which was confirmed when re-introduced into Arabidopsis (Kakimoto, Science 274: 982-5, 1996). In a more recent report, activation T-DNA tagging and screening plants for an early flowering phenotype led to the isolation of the FT gene (Kardailsky et al., Science 286: 1962-1965, 1999).
Variations of the activation tagging technique include the use of the Agrobacterium gene 5 promoter (pg5), which is active only in proliferating cells and must insert directly adjacent to a plant gene in order to influence its expression, using, e.g., the nos promoter/hpt selection cassette (pCVHPT), originally described in Koncz et al., Proc Natl Acad Sci USA 86(21): 8467-8471, 1989. Another form of activation tagging utilizes a modified Ds transposon carrying the CaMV 35S promoter and a nos::hpt selection cassette (Wilson et al., Plant Cell 8: 659-671, 1996). The modified Ds element is inserted into an antibiotic resistance cassette within a binary vector expression construct. Once introduced into Arabidopsis, the transposed Ds element (via the resident 35S promoter) is able to upregulate adjacent plant genes resulting in dominant gain-of-function mutations (Schaffer et al., Cell 93: 1219-1229, 1998; Wilson et al., Plant Cell 8: 659-671, 1996). Activation tagging vectors have been developed that are useful for screening tens of thousands of transformed plants for morphological phenotypes (Weigel et al., Plant Physiology, 122: 1003-1013, 2000).